(a) Field of the Invention
The invention relates to an isolated and characterized neuron promoter serving as a neurogenesis and neuronal differentiation marker and for use in transgenic mammals.
(b) Description of Prior Art
Neurons are highly polarized cells whose most obvious feature is their extensive system of axons and dendrites. This complex morphology is elaborated during embryogenesis and early postnatal life in mice, and likely involves a complex interplay between extrinsic influences and intrinsic neuronal genetic mechanisms. New morphological growth also occurs in the mature nervous system in response to neural trauma or pathology (Brown, 1984, TINS, 1:10-14), and may be an ongoing phenomena in the normal, mature animal (Purves et al., 1986, J. Neurosci., 6:1051-1060). The biological importance of such structural plasticity largely derives from the fact that, neurons of the mature nervous system neither develop de novo nor migrate to new positions in substantial numbers. Thus, the growth and remodeling of mature neurons provides the nervous system with one potential cellular mechanism for responding to ongoing physiological and/or pathological phenomena. The molecular mechanisms underlying morphological growth, and the extraneuronal cues that regulate it remain largely undefined.
Microtubules, which are assembled from .alpha.- and .beta.tubulins, comprise the major cytoskeletal component of growing neurites (Daniels, 1972, J. Cell. Biol., 53:164-176). In mammals, at least 6 different .alpha.-tubulin genes (Villasante et al., 1986, Mol. Cell. Biol., 6:2409-2419) and 5 different .beta.-tubulin genes (Wang et al., 1986, J. Cell. Biol., 103:1903-1910) are expressed in neural and nonneural tissues at various times during development. It has been previously demonstrated that, of two .alpha.-tubulin genes known to be expressed in the embryonic nervous system of the rat, one, termed T.alpha.1, is abundantly expressed in developing neurons during morphological growth, while a second, termed T26, is constitutively expressed in neurons and nonneuronal cells (Miller et al., 1987, J. Cell. Biol., 105:3065-3073). Moreover, it is likely that the T.alpha.1 isotype is incorporated in the majority of neuronal microtubules during development, since T.alpha.1 mRNA comprises 1-2% of the total mRNA, and &gt;95% of the total .alpha.-tubulin mRNA in the embryonic nervous system.
Expression of T.alpha.1 .alpha.-tubulin mRNA is also correlated with the growth of mature neurons. Following axotomy of motor (Miller et al., 1989, J. Neurosci., 9:1452-1463), and sympathetic (Mathew and Miller, 1993, Dev. Biol., 158:467-474; Wu et al., 1993, Dev. Biol., 158:456-466) neurons, T.alpha.1 mRNA increases rapidly, peaks at 3 to 7 days postlesion, and decreases to control levels following target reinnervation. If regeneration is unsuccessful, as with central nervous system (CNS) neurons (Tetzlaff et al., 1991, J. Neurosci., 11:2528-2544), or with transection of peripheral neurons (Miller et al., 1989, J. Neurosci., 9:1452-1463), T.alpha.1 mRNA levels remain elevated. These increases appear to be due, to a great extent, to loss of repressive homeostatic signals arising from the non-terminal axon (Mathew and Miller, 1990, Dev. Biol., 141:84-92). T.alpha.1 .alpha.-tubulin mRNA levels are also increased during collateral sprouting of adult sympathetic neurons (Mathew and Miller, 1990, Dev. Biol., 141:84-92), presumably in response to increased target-derived nerve growth factor (NGF), since exogenous NGF can increase T.alpha.1 .alpha.-tubulin mRNA levels in these neurons both in vivo and in culture (Ma et al., 1992, J. Cell. Biol., 117:135-141). Thus, expression of T.alpha.1 mRNA is high during developmental growth, is down-regulated as a function of neuronal maturation, and is then increased in response to extrinsic cues that regulate the growth of mature neurons.
The functional role of these high levels of expression of one particular .alpha.-tubulin isotype during neuronal growth remains speculative. The rat T.alpha.1, human b.alpha.1, and mouse M.alpha.1 .alpha.-tubulin mRNAs all encode identical proteins and share 82% homology in their 3' noncoding regions (Lemischka et al., 1981, J. Mol. Biol., 150:101-120). Although the rat T26 mRNA sequence is not complete (Ginzburg et al., 1986, Nucleic Acid Res., 9:2691-2697), the defined amino acid sequence is identical to that shared by human k.alpha.1 (Cowan et al., 1983, Mol. Cell. Biol., 3:1738-1745), and mouse M.alpha.1 (Lewis et al., 1985, J. Cell. Biol., 101:852-861), and the 3' noncoding regions of the three mRNAs share 72% homology. The k.alpha.1 and M.alpha.2 isotypes differ from the T.alpha.1, b.alpha.1, and M.alpha.1 isotypes by virtue of a single amino acid difference (serine to glycine) at residue 232. On the basis of these sequence similarities, it was previously speculated that T.alpha.1 and T26 mRNAs encode functionally equivalent proteins, and that the T.alpha.1 gene itself is specialized to produce a large pool of .alpha.-tubulin monomers when this is in high demand for neurite extension.
In the International Patent Application No. WO/9307280, there is disclosed a human astrocyte-specific gene expression system capable of regulating astrocyte-specific transcription of the human gene for glial fibrillary acidic protein (GFAP). This gene expression system can be used to express gene products de novo or to increase their expression in astrocytes and transgenic animals. This gene expression system can also be used to create transgenic animal models for evaluating Alzheimer's diseases.
It would be highly desirable to be provided with an isolated endogenous neuron promoter expressed during growth of both developing and mature neurons.
It would be highly desirable to be provided with a fusion gene of such a promoter linked to a marker gene wherein such a fusion gene would induce abundant expression of the marker during neurogenesis and neuronal differentiation.
It would be highly desirable to be provided with a screening assay for pharmaceutical agents that promote neuronal survival.
It would be highly desirable to be provided with a neuron promoter for targeting gene therapy to injured neurons.
It would be highly desirable to be provided with transgenic neurons expressing a neuronal promoter for use in transplantation studies.
It would be highly desirable to be provided with an in vivo model for toxicology studies.